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1.
Mol Biochem Parasitol ; 244: 111375, 2021 07.
Article in English | MEDLINE | ID: mdl-34023299

ABSTRACT

Malaria parasites exhibit a complex lifecycle, requiring extensive asexual replication in the liver and blood of the vertebrate host, and in the haemocoel of the insect vector. Yet, they must also undergo a single round of sexual reproduction, which occurs in the vector's midgut upon uptake of a blood meal. Sexual reproduction is obligate for infection of the vector and thus, is essential for onwards transmission to new hosts. Sex in malaria parasites involves several bottlenecks in parasite number, making the stages involved attractive targets for blocking disease transmission. Malaria parasites have evolved a suite of adaptations ("strategies") to maximise the success of sexual reproduction and transmission, which could undermine transmission-blocking interventions. Yet, understanding parasite strategies may also reveal novel opportunities for such interventions. Here, we outline how evolutionary and ecological theories, developed to explain reproductive strategies in multicellular taxa, can be applied to explain two reproductive strategies (conversion rate and sex ratio) expressed by malaria parasites within the vertebrate host.


Subject(s)
Gametogenesis , Life Cycle Stages/genetics , Malaria/parasitology , Plasmodium berghei/growth & development , Plasmodium chabaudi/growth & development , Plasmodium falciparum/growth & development , Plasmodium knowlesi/growth & development , Animals , Biological Coevolution , Culicidae/parasitology , Erythrocytes/parasitology , Female , Host-Parasite Interactions/genetics , Humans , Insect Vectors/parasitology , Liver/parasitology , Malaria/transmission , Male , Plasmodium berghei/genetics , Plasmodium berghei/metabolism , Plasmodium chabaudi/genetics , Plasmodium chabaudi/metabolism , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Plasmodium knowlesi/genetics , Plasmodium knowlesi/metabolism , Reproduction, Asexual , Sex Ratio
2.
Nat Commun ; 11(1): 2763, 2020 06 02.
Article in English | MEDLINE | ID: mdl-32488076

ABSTRACT

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 h suggesting a circadian basis, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches utilizing rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit daily rhythms in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 6% of Plasmodium falciparum genes show 24 h rhythms in expression under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a 24 h transcriptional rhythm and disrupting it in rodent malaria parasites shortens the IDC by 2-3 h; (v) Multiple processes including DNA replication, and the ubiquitin and proteasome pathways, are affected by loss of coordination with host rhythms and by disruption of SR10. Our results reveal malaria parasites are at least partly responsible for scheduling the IDC and coordinating their development with host daily rhythms.


Subject(s)
Circadian Rhythm/physiology , Erythropoiesis/physiology , Host-Parasite Interactions/physiology , Malaria/metabolism , Protozoan Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Secologanin Tryptamine Alkaloids/metabolism , Animals , Caenorhabditis elegans Proteins , Disease Models, Animal , Female , Gene Expression , Host-Parasite Interactions/genetics , Humans , Malaria/parasitology , Mice , Mice, Knockout , Plasmodium chabaudi/genetics , Plasmodium chabaudi/growth & development , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Protozoan Proteins/genetics , Receptors, G-Protein-Coupled/genetics , Rodentia , Transcriptome
3.
Malar J ; 19(1): 17, 2020 Jan 14.
Article in English | MEDLINE | ID: mdl-31937300

ABSTRACT

BACKGROUND: The intraerythrocytic development cycle (IDC) of the rodent malaria Plasmodium chabaudi is coordinated with host circadian rhythms. When this coordination is disrupted, parasites suffer a 50% reduction in both asexual stages and sexual stage gametocytes over the acute phase of infection. Reduced gametocyte density may not simply follow from a loss of asexuals because investment into gametocytes ("conversion rate") is a plastic trait; furthermore, the densities of both asexuals and gametocytes are highly dynamic during infection. Hence, the reasons for the reduction of gametocytes in infections that are out-of-synch with host circadian rhythms remain unclear. Here, two explanations are tested: first, whether out-of-synch parasites reduce their conversion rate to prioritize asexual replication via reproductive restraint; second, whether out-of-synch gametocytes experience elevated clearance by the host's circadian immune responses. METHODS: First, conversion rate data were analysed from a previous experiment comparing infections of P. chabaudi that were in-synch or 12 h out-of-synch with host circadian rhythms. Second, three new experiments examined whether the inflammatory cytokine TNF varies in its gametocytocidal efficacy according to host time-of-day and gametocyte age. RESULTS: There was no evidence that parasites reduce conversion or that their gametocytes become more vulnerable to TNF when out-of-synch with host circadian rhythms. CONCLUSIONS: The factors causing the reduction of gametocytes in out-of-synch infections remain mysterious. Candidates for future investigation include alternative rhythmic factors involved in innate immune responses and the rhythmicity in essential resources required for gametocyte development. Explaining why it matters for gametocytes to be synchronized to host circadian rhythms might suggest novel approaches to blocking transmission.


Subject(s)
Circadian Rhythm , Erythrocytes/parasitology , Malaria/parasitology , Plasmodium chabaudi/physiology , Tumor Necrosis Factor-alpha/administration & dosage , Animals , Circadian Rhythm/immunology , Female , Flow Cytometry , Gametogenesis/physiology , Linear Models , Malaria/blood , Malaria/immunology , Male , Merozoites/physiology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Plasmodium chabaudi/genetics , Plasmodium chabaudi/growth & development , Plasmodium chabaudi/immunology , Random Allocation , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/immunology
4.
Infect Immun ; 87(1)2019 01.
Article in English | MEDLINE | ID: mdl-30323025

ABSTRACT

Naturally acquired immunity to malaria is robust and protective against all strains of the same species of Plasmodium This develops as a result of repeated natural infection, taking several years to develop. Evidence suggests that apoptosis of immune lymphocytes due to uncontrolled parasite growth contributes to the slow acquisition of immunity. To hasten and augment the development of natural immunity, we studied controlled infection immunization (CII) using low-dose exposure to different parasite species (Plasmodium chabaudi, P. yoelii, or P. falciparum) in two rodent systems (BALB/c and C57BL/6 mice) and in human volunteers, with drug therapy commencing at the time of initiation of infection. CIIs with infected erythrocytes and in conjunction with doxycycline or azithromycin, which are delayed death drugs targeting the parasite's apicoplast, allowed extended exposure to parasites at low levels. In turn, this induced strong protection against homologous challenge in all immunized mice. We show that P. chabaudi/P. yoelii infection initiated at the commencement of doxycycline therapy leads to cellular or antibody-mediated protective immune responses in mice, with a broad Th1 cytokine response providing the best correlate of protection against homologous and heterologous species of PlasmodiumP. falciparum CII with doxycycline was additionally tested in a pilot clinical study (n = 4) and was found to be well tolerated and immunogenic, with immunological studies primarily detecting increased cell-associated immune responses. Furthermore, we report that a single dose of the longer-acting drug, azithromycin, given to mice (n = 5) as a single subcutaneous treatment at the initiation of infection controlled P. yoelii infection and protected all mice against subsequent challenge.


Subject(s)
Antimalarials/administration & dosage , Malaria/drug therapy , Malaria/immunology , Plasmodium chabaudi/immunology , Plasmodium falciparum/immunology , Plasmodium yoelii/immunology , Vaccination/methods , Adaptive Immunity , Animals , Azithromycin/administration & dosage , Cytokines/metabolism , Disease Models, Animal , Doxycycline/administration & dosage , Female , Humans , Malaria/prevention & control , Malaria, Falciparum , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Plasmodium chabaudi/growth & development , Plasmodium falciparum/growth & development , Plasmodium yoelii/growth & development , Th1 Cells/immunology , Young Adult
5.
PLoS Pathog ; 14(2): e1006900, 2018 02.
Article in English | MEDLINE | ID: mdl-29481559

ABSTRACT

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the host's peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the host's peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new arena for studying host-parasite-vector coevolution and has broad implications for applied bioscience.


Subject(s)
Circadian Rhythm/physiology , Feeding Behavior/physiology , Host-Parasite Interactions/physiology , Malaria/parasitology , Animals , Blood Glucose/analysis , Gastrointestinal Microbiome/physiology , Homeostasis , Malaria/blood , Malaria/physiopathology , Male , Mice , Plasmodium chabaudi/growth & development , Plasmodium chabaudi/physiology
6.
Infect Immun ; 85(12)2017 12.
Article in English | MEDLINE | ID: mdl-28893916

ABSTRACT

Epidemiological observations have linked increased host iron with malaria susceptibility, and perturbed iron handling has been hypothesized to contribute to the potentially life-threatening anemia that may accompany blood-stage malaria infection. To improve our understanding of these relationships, we examined the pathways involved in regulation of the master controller of iron metabolism, the hormone hepcidin, in malaria infection. We show that hepcidin upregulation in Plasmodium berghei murine malaria infection was accompanied by changes in expression of bone morphogenetic protein (BMP)/sons of mothers against decapentaplegic (SMAD) pathway target genes, a key pathway involved in hepcidin regulation. We therefore investigated known agonists of the BMP/SMAD pathway and found that Bmp gene expression was not increased in infection. In contrast, activin B, which can signal through the BMP/SMAD pathway and has been associated with increased hepcidin during inflammation, was upregulated in the livers of Plasmodium berghei-infected mice; hepatic activin B was also upregulated at peak parasitemia during infection with Plasmodium chabaudi Concentrations of the closely related protein activin A increased in parallel with hepcidin in serum from malaria-naive volunteers infected in controlled human malaria infection (CHMI) clinical trials. However, antibody-mediated neutralization of activin activity during murine malaria infection did not affect hepcidin expression, suggesting that these proteins do not stimulate hepcidin upregulation directly. In conclusion, we present evidence that the BMP/SMAD signaling pathway is perturbed in malaria infection but that activins, although raised in malaria infection, may not have a critical role in hepcidin upregulation in this setting.


Subject(s)
Activins/metabolism , Hepcidins/metabolism , Malaria/pathology , Plasmodium berghei/growth & development , Plasmodium chabaudi/growth & development , Animals , Disease Models, Animal , Gene Expression Regulation , Humans , Mice
7.
PLoS One ; 12(6): e0176533, 2017.
Article in English | MEDLINE | ID: mdl-28604779

ABSTRACT

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.


Subject(s)
Erythrocytes/parasitology , Life Cycle Stages , Lysine/metabolism , Malaria/veterinary , Plasmodium chabaudi/growth & development , Plasmodium chabaudi/metabolism , Rodent Diseases/parasitology , Ubiquitination , Alternative Splicing , Animals , Gene Expression Regulation , Mass Spectrometry , Plasmodium chabaudi/genetics , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitination/genetics
8.
Parasitol Int ; 66(2): 89-99, 2017 Apr.
Article in English | MEDLINE | ID: mdl-27919743

ABSTRACT

Recent evidence suggests that 1α,25-dihydroxyvitamin D3 (calcitriol, VD3), the active form of vitamin D (VD), can inhibit the proliferation of microorganisms. In the present study, we conducted in vitro experiments and utilized in vivo murine models to investigate the antimalarial activity of VD3 and its analog, 22-oxacalcitriol (22-OCT), which was designed to cause less hypercalcemia than VD3. VD3 and 22-OCT treatments effectively resolved a Plasmodium chabaudi (Pc) infection in wild-type mice. Reduced parasitemia was observed during the acute phase of infection in the presence of VD3 and 22-OCT, followed by a delayed peak during the chronic stage of infection. Some anti-Pc activity was observed in VD receptor knockout (KO) mice. VD3 and 22-OCT also completely inhibited the proliferation of P. falciparum (Pf) in human red blood cells in vitro. Plasma levels of interferon (IFN)-γ in VD3-treated B10 and B6 mice were lower than those in vehicle-treated animals, and VD3 resolved a Pc infection in IFN-γ-KO mice, which greatly improved survival. These data suggest that the protective effects of VD3 are elicited through an IFN-γ-independent mechanism. Effective antiplasmodial doses of VD3 and 22-OCT resulted in a loss of body weight in mice. This loss in body weight occurred concomitantly with the development of hypercalcemia. Zoledronic acid partially attenuated VD3-induced hypercalcemia and abrogated the antiparasitic effects of VD3. This study highlights a potential therapeutic role for VD3 in the treatment of malarial infections and shows that hypercalcemia is excellent indicator of the antiplasmodial activity of VD3.


Subject(s)
Antimalarials/pharmacology , Calcitriol/analogs & derivatives , Cholecalciferol/pharmacology , Malaria/drug therapy , Plasmodium chabaudi/drug effects , Acute Disease/therapy , Animals , Antimalarials/therapeutic use , Body Weight/drug effects , Calcitriol/pharmacology , Calcitriol/therapeutic use , Cholecalciferol/administration & dosage , Chronic Disease/drug therapy , Diphosphonates/pharmacology , Diphosphonates/therapeutic use , Erythrocytes/drug effects , Erythrocytes/parasitology , Humans , Hypercalcemia/blood , Hypercalcemia/drug therapy , Imidazoles/pharmacology , Imidazoles/therapeutic use , Interferon-gamma/blood , Interferon-gamma/deficiency , Malaria/parasitology , Mice , Mice, Inbred BALB C , Mice, Knockout , Parasitemia/drug therapy , Parasitemia/immunology , Parasitemia/parasitology , Plasmodium chabaudi/growth & development , Plasmodium chabaudi/immunology , Receptors, Calcitriol/deficiency , Zoledronic Acid
9.
Malar J ; 15(1): 428, 2016 08 24.
Article in English | MEDLINE | ID: mdl-27557867

ABSTRACT

BACKGROUND: Cerebral malaria is one of the most severe complications of Plasmodium falciparum infection and occurs mostly in young African children. This syndrome results from a combination of high levels of parasitaemia and inflammation. Although parasite sequestration in the brain is a feature of the human syndrome, sequestering strains do not uniformly cause severe malaria, suggesting interplay with other factors. Host genetic factors such as mutations in the promoters of the cytokines IL-10 and TNF are also clearly linked to severe disease. Plasmodium chabaudi, a rodent malaria parasite, leads to mild illness in wildtype animals. However, IL-10(-/-) mice respond to parasite with increased levels of pro-inflammatory cytokines IFN-γ and TNF, leading to lethal disease in the absence of sequestration in the brain. These mice also exhibit cerebral symptoms including gross cerebral oedema and haemorrhage, allowing study of these critical features of disease without the influence of sequestration. METHODS: The neurological consequences of P. chabaudi infection were investigated by performing a general behavioural screen (SHIRPA). The immune cell populations found in the brain during infection were also analysed using flow cytometry and confocal microscopy. RESULTS: IL-10(-/-) mice suffer significant declines in behavioural and physical capacities during infection compared to wildtype. In addition, grip strength and pain sensitivity were affected, suggestive of neurological involvement. Several immune cell populations were identified in the perfused brain on day 7 post-infection, suggesting that they are tightly adherent to the vascular endothelium, or potentially located within the brain parenchyma. There was an increase in both inflammatory monocyte and resident macrophage (CD11b(hi), CD45(+), MHCII(+), Ly6C(+/-)) numbers in IL-10(-/-) compared to wildtype animals. In addition, the activation state of all monocytes and microglia (CD11b(int), CD45(-), MHC-II(+)) were increased. T cells making IFN-γ were also identified in the brain, but were localized within the vasculature, and not the parenchyma. CONCLUSIONS: These studies demonstrate exacerbated neuroinflammation concurrent with development of behavioural symptoms in P. chabaudi infection of IL-10(-/-) animals.


Subject(s)
Behavior, Animal , Inflammation/pathology , Interleukin-10/deficiency , Malaria, Cerebral/complications , Malaria, Cerebral/pathology , Mental Disorders/etiology , Plasmodium chabaudi/growth & development , Animals , Brain/pathology , Disease Models, Animal , Female , Flow Cytometry , Humans , Leukocytes/immunology , Malaria, Cerebral/parasitology , Male , Mice, Inbred C57BL , Microscopy, Confocal
10.
J Struct Biol ; 194(2): 171-9, 2016 May.
Article in English | MEDLINE | ID: mdl-26882843

ABSTRACT

In the course of their intraerythrocytic development, malaria parasites incorporate and degrade massive amounts of the host cell cytoplasm. This mechanism is essential for parasite development and represents a physiological step used as target for many antimalarial drugs; nevertheless, the fine mechanisms underlying these processes in Plasmodium species are still under discussion. Here, we studied the events of hemoglobin uptake and hemozoin nucleation in the different stages of the intraerythrocytic cycle of the murine malaria parasite Plasmodium chabaudi using transmission electron tomography of cryofixed and freeze-substituted cells. The results showed that hemoglobin uptake in P. chabaudi starts at the early ring stage and is present in all developmental stages, including the schizont stage. Hemozoin nucleation occurs near the membrane of small food vacuoles. At the trophozoite stage, food vacuoles are found closely localized to cytostomal tubes and mitochondria, whereas in the schizont stage, we observed a large food vacuole located in the central portion of the parasite. Taken together, these results provide new insights into the mechanisms of hemoglobin uptake and degradation in rodent malaria parasites.


Subject(s)
Hemeproteins/metabolism , Hemoglobins/metabolism , Life Cycle Stages/physiology , Plasmodium chabaudi/metabolism , Vacuoles/metabolism , Animals , Biological Transport , Electron Microscope Tomography , Erythrocytes/metabolism , Erythrocytes/parasitology , Erythrocytes/ultrastructure , Hemeproteins/ultrastructure , Hemoglobins/ultrastructure , Image Processing, Computer-Assisted , Malaria/parasitology , Male , Mice , Plasmodium chabaudi/growth & development , Plasmodium chabaudi/ultrastructure , Proteolysis , Vacuoles/ultrastructure
11.
Malar J ; 12: 440, 2013 Dec 06.
Article in English | MEDLINE | ID: mdl-24314024

ABSTRACT

BACKGROUND: A series of elegant experiments was recently published which demonstrated that transmission of malaria parasites through mosquitoes elicited an attenuated growth phenotype, whereby infections grew more slowly and reached peak parasitaemia at least five-fold lower than parasites which had not been mosquito transmitted. To assess the implications of these results it is essential to understand whether the attenuated infection phenotype is a general phenomenon across parasites genotypes and conditions. METHODS: Using previously published data, the impact of mosquito transmission on parasite growth rates and virulence of six Plasmodium chabaudi lines was analysed. RESULTS: The effect of mosquito transmission varied among strains, but did not lead to pronounced or consistent reductions in parasite growth rate. CONCLUSIONS: Mosquito-induced attenuated growth phenotype is sensitive to experimental conditions.


Subject(s)
Culicidae/parasitology , Malaria/parasitology , Plasmodium chabaudi/pathogenicity , Animals , Biomedical Research , Malaria/transmission , Mice , Phenotype , Plasmodium chabaudi/genetics , Plasmodium chabaudi/growth & development , Research Design , Virulence
12.
Malar J ; 12: 372, 2013 Oct 26.
Article in English | MEDLINE | ID: mdl-24160251

ABSTRACT

BACKGROUND: In the blood, the synchronous malaria parasite, Plasmodium chabaudi, exhibits a cell-cycle rhythm of approximately 24 hours in which transitions between developmental stages occur at particular times of day in the rodent host. Previous experiments reveal that when the timing of the parasite's cell-cycle rhythm is perturbed relative to the circadian rhythm of the host, parasites suffer a (~50%) reduction in asexual stages and gametocytes. Why it matters for parasites to have developmental schedules in synchronization with the host's rhythm is unknown. The experiment presented here investigates this issue by: (a) validating that the performance of P. chabaudi is negatively affected by mismatch to the host circadian rhythm; (b) testing whether the effect of mismatch depends on the route of infection or the developmental stage of inoculated parasites; and, (c) examining whether the costs of mismatch are due to challenges encountered upon initial infection and/or due to ongoing circadian host processes operating during infection. METHODS: The experiment simultaneously perturbed the time of day infections were initiated, the stage of parasite inoculated, and the route of infection. The performance of parasites during the growth phase of infections was compared across the cross-factored treatment groups (i e, all combinations of treatments were represented). RESULTS: The data show that mismatch to host rhythms is costly for parasites, reveal that this phenomenon does not depend on the developmental stage of parasites nor the route of infection, and suggest that processes operating at the initial stages of infection are responsible for the costs of mismatch. Furthermore, mismatched parasites are less virulent, in that they cause less anaemia to their hosts. CONCLUSION: It is beneficial for parasites to be in synchronization with their host's rhythm, regardless of the route of infection or the parasite stage inoculated. Given that arrested cell-cycle development (quiescence) is implicated in tolerance to drugs, understanding how parasite schedules are established and maintained in the blood is important.


Subject(s)
Circadian Rhythm , Malaria/parasitology , Plasmodium chabaudi/growth & development , Rodentia/physiology , Rodentia/parasitology , Animals , Male , Mice
13.
PLoS Pathog ; 9(9): e1003578, 2013 Sep.
Article in English | MEDLINE | ID: mdl-24068922

ABSTRACT

Drug resistant pathogens are one of the key public health challenges of the 21st century. There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold), without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.


Subject(s)
Antimalarials/administration & dosage , Drug Resistance , Malaria/drug therapy , Models, Biological , Plasmodium chabaudi/drug effects , Selection, Genetic/drug effects , Animals , Antimalarials/adverse effects , Antimalarials/pharmacology , Antimalarials/therapeutic use , Clone Cells , Dose-Response Relationship, Drug , Erythrocytes/drug effects , Erythrocytes/parasitology , Female , Malaria/blood , Malaria/parasitology , Mice, Inbred C57BL , Parasite Egg Count , Plasmodium chabaudi/genetics , Plasmodium chabaudi/growth & development , Plasmodium chabaudi/pathogenicity , Pyrimethamine/administration & dosage , Pyrimethamine/adverse effects , Pyrimethamine/pharmacology , Pyrimethamine/therapeutic use , Virulence/drug effects
14.
Parasitol Res ; 112(11): 3757-70, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23949311

ABSTRACT

Epigenetic reprogramming of host genes via DNA methylation is increasingly recognized as critical for the outcome of diverse infectious diseases, but information for malaria is not yet available. Here, we investigate the effect of blood-stage malaria of Plasmodium chabaudi on the DNA methylation status of host gene promoters on a genome-wide scale using methylated DNA immunoprecipitation and Nimblegen microarrays containing 2,000 bp oligonucleotide features that were split into -1,500 to -500 bp Ups promoters and -500 to +500 bp Cor promoters, relative to the transcription site, for evaluation of differential DNA methylation. Gene expression was analyzed by Agilent and Affymetrix microarray technology. Challenging of female C57BL/6 mice with 10(6) P. chabaudi-infected erythrocytes resulted in a self-healing outcome of infections with peak parasitemia on day 8 p.i. These infections induced organ-specific modifications of DNA methylation of gene promoters. Among the 17,354 features on Nimblegen arrays, only seven gene promoters were identified to be hypermethylated in the spleen, whereas the liver exhibited 109 hyper- and 67 hypomethylated promoters at peak parasitemia in comparison with non-infected mice. Among the identified genes with differentially methylated Cor-promoters, only the 7 genes Pigr, Ncf1, Klkb1, Emr1, Ndufb11, and Tlr6 in the liver and Apol6 in the spleen were detected to have significantly changed their expression. Remarkably, the Cor promoter of the toll-like receptor Tlr6 became hypomethylated and Tlr6 expression increased by 3.4-fold during infection. Concomitantly, the Ups promoter of the Tlr1 was hypermethylated, but Tlr1 expression also increased by 11.3-fold. TLR6 and TLR1 are known as auxillary receptors to form heterodimers with TLR2 in plasma membranes of macrophages, which recognize different pathogen-associated molecular patterns (PAMPs), as, e.g., intact 3-acyl and sn-2-lyso-acyl glycosylphosphatidylinositols of P. falciparum, respectively. Our data suggest therefore that malaria-induced epigenetic fine-tuning of Tlr6 and Tlr1 through DNA methylation of their gene promoters in the liver is critically important for initial recognition of PAMPs and, thus, for the final self-healing outcome of blood-stage infections with P. chabaudi malaria.


Subject(s)
Host-Pathogen Interactions , Liver/parasitology , Plasmodium chabaudi/growth & development , Promoter Regions, Genetic , Spleen/parasitology , Toll-Like Receptor 1/genetics , Toll-Like Receptor 6/genetics , Animals , Chromatin Immunoprecipitation , DNA Methylation , Epigenesis, Genetic , Female , Gene Expression Profiling , Gene Expression Regulation , Liver/pathology , Mice , Mice, Inbred C57BL , Microarray Analysis , Plasmodium chabaudi/enzymology , Spleen/pathology
15.
Exp Parasitol ; 135(1): 166-74, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23830988

ABSTRACT

Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model.


Subject(s)
Antibodies, Protozoan/biosynthesis , Cysteine Proteases/immunology , Malaria Vaccines , Malaria/prevention & control , Plasmodium chabaudi/enzymology , Plasmodium chabaudi/immunology , Animals , Anopheles/parasitology , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antibodies, Protozoan/immunology , Cysteine Proteases/analysis , Cysteine Proteases/genetics , Cytokines/metabolism , Disease Models, Animal , Erythrocytes/parasitology , Female , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , Plasmodium berghei/physiology , Plasmodium chabaudi/growth & development , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Vaccines, Synthetic
16.
Nature ; 498(7453): 228-31, 2013 Jun 13.
Article in English | MEDLINE | ID: mdl-23719378

ABSTRACT

Defining mechanisms by which Plasmodium virulence is regulated is central to understanding the pathogenesis of human malaria. Serial blood passage of Plasmodium through rodents, primates or humans increases parasite virulence, suggesting that vector transmission regulates Plasmodium virulence within the mammalian host. In agreement, disease severity can be modified by vector transmission, which is assumed to 'reset' Plasmodium to its original character. However, direct evidence that vector transmission regulates Plasmodium virulence is lacking. Here we use mosquito transmission of serially blood passaged (SBP) Plasmodium chabaudi chabaudi to interrogate regulation of parasite virulence. Analysis of SBP P. c. chabaudi before and after mosquito transmission demonstrates that vector transmission intrinsically modifies the asexual blood-stage parasite, which in turn modifies the elicited mammalian immune response, which in turn attenuates parasite growth and associated pathology. Attenuated parasite virulence associates with modified expression of the pir multi-gene family. Vector transmission of Plasmodium therefore regulates gene expression of probable variant antigens in the erythrocytic cycle, modifies the elicited mammalian immune response, and thus regulates parasite virulence. These results place the mosquito at the centre of our efforts to dissect mechanisms of protective immunity to malaria for the development of an effective vaccine.


Subject(s)
Culicidae/parasitology , Host-Parasite Interactions/immunology , Insect Vectors/parasitology , Plasmodium chabaudi/immunology , Plasmodium chabaudi/pathogenicity , Animals , Erythrocytes/parasitology , Malaria/immunology , Malaria/parasitology , Malaria/transmission , Malaria Vaccines/immunology , Mice , Mice, Inbred C57BL , Plasmodium chabaudi/growth & development , Plasmodium chabaudi/isolation & purification , Serial Passage , Virulence/immunology
17.
Infect Genet Evol ; 14: 340-6, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23318648

ABSTRACT

If drug-resistant malaria mutants are less fit than sensitive forms, they will wane over time when active drug pressure is removed and the overall sensitivity to the drug may be restored. However, most studies addressing this issue have been largely retrospective. Here, we undertook a predictive study, using mutant rodent malaria parasites resistant to the Artemisinin combination treatment (ACT) version of artesunate+mefloquine (ATN+MF) to gain insights about their ability to compete with ATN+MF-sensitive forms in untreated hosts. Previously, Plasmodium chabaudi parasites resistant to ATN+MF were selected in vivo through prolonged passaging in mice under increasing doses of the two drugs, and shown to harbour duplication of the mdr1 gene. Here, the resistant parasite, AS-ATNMF1, was mixed with its progenitor AS-ATN in different proportions and each mixture was injected into mice that were left untreated. Absolute percentage parasitaemias and the proportion of each parasite were then monitored by microscopy and proportional sequencing, respectively, every two days for a period of 14days. AS-ATNMF1 outperformed its progenitor AS-ATN over the whole sampling period regardless of the relative starting proportion of each parasite clone. In order to assess if consecutive sub-inoculations could have been responsible for the apparent fitness gain of the resistant parasite, its growth was compared to that of AS-ATN27P, a parasite which was passaged the same number of times as AS-ATNMF1, but left untreated. Although small fluctuations in the proportion of each parasite were observed through time, the relative abundance of each on the last day of sampling (Day 14) was virtually identical to that of the starting inoculum. We conclude that there is no fitness cost associated with MDR1-associated ATN+MF resistance in vivo. These observations offer the first insights about the within-host dynamics between ACT-resistant and -sensitive parasites in absence of drug pressure.


Subject(s)
Antimalarials/pharmacology , Artemisinins/pharmacology , Drug Resistance/genetics , Multidrug Resistance-Associated Proteins/genetics , Plasmodium chabaudi/drug effects , Plasmodium chabaudi/genetics , Protozoan Proteins/genetics , Animals , Artesunate , Disease Models, Animal , Host-Pathogen Interactions , Male , Mice , Parasitemia/drug therapy , Parasitemia/parasitology , Plasmodium chabaudi/growth & development
18.
PLoS One ; 7(6): e38999, 2012.
Article in English | MEDLINE | ID: mdl-22723917

ABSTRACT

The blood stage of the plasmodium parasite life cycle is responsible for the clinical symptoms of malaria. Epidemiological studies have identified coincidental malarial endemicity and multiple red blood cell (RBC) disorders. Many RBC disorders result from mutations in genes encoding cytoskeletal proteins and these are associated with increased protection against malarial infections. However the mechanisms underpinning these genetic, host responses remain obscure. We have performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen and have identified a novel dominant (haploinsufficient) mutation in the Ank-1 gene (Ank1(MRI23420)) of mice displaying hereditary spherocytosis (HS). Female mice, heterozygous for the Ank-1 mutation showed increased survival to infection by Plasmodium chabaudi adami DS with a concomitant 30% decrease in parasitemia compared to wild-type, isogenic mice (wt). A comparative in vivo red cell invasion and parasite growth assay showed a RBC-autonomous effect characterised by decreased proportion of infected heterozygous RBCs. Within approximately 6-8 hours post-invasion, TUNEL staining of intraerythrocytic parasites, showed a significant increase in dead parasites in heterozygotes. This was especially notable at the ring and trophozoite stages in the blood of infected heterozygous mutant mice compared to wt (p<0.05). We conclude that increased malaria resistance due to ankyrin-1 deficiency is caused by the intraerythrocytic death of P. chabaudi parasites.


Subject(s)
Ankyrins/genetics , Erythrocytes/parasitology , Ethylnitrosourea/adverse effects , Malaria/parasitology , Mutation/drug effects , Plasmodium chabaudi/growth & development , Alleles , Amino Acid Sequence , Animals , Ankyrins/metabolism , Base Sequence , Erythrocytes/metabolism , Erythrocytes/ultrastructure , Female , Heterozygote , Malaria/mortality , Male , Mice , Molecular Sequence Data , Phenotype , Spherocytosis, Hereditary/metabolism
19.
BMC Genomics ; 13: 125, 2012 Mar 29.
Article in English | MEDLINE | ID: mdl-22458863

ABSTRACT

BACKGROUND: The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due to antigenic variation. In this model, pir genes are called cirs and may be involved in this mechanism, allowing evasion of host immune responses. In order to fully understand the role(s) of CIR proteins during P. chabaudi infection, a detailed characterization of the cir gene family was required. RESULTS: The cir repertoire was annotated and a detailed bioinformatic characterization of the encoded CIR proteins was performed. Two major sub-families were identified, which have been named A and B. Members of each sub-family displayed different amino acid motifs, and were thus predicted to have undergone functional divergence. In addition, the expression of the entire cir repertoire was analyzed via RNA sequencing and microarray. Up to 40% of the cir gene repertoire was expressed in the parasite population during infection, and dominant cir transcripts could be identified. In addition, some differences were observed in the pattern of expression between the cir subgroups at the peak of P. chabaudi infection. Finally, specific cir genes were expressed at different time points during asexual blood stages. CONCLUSIONS: In conclusion, the large number of cir genes and their expression throughout the intraerythrocytic cycle of development indicates that CIR proteins are likely to be important for parasite survival. In particular, the detection of dominant cir transcripts at the peak of P. chabaudi infection supports the idea that CIR proteins are expressed, and could perform important functions in the biology of this parasite. Further application of the methodologies described here may allow the elucidation of CIR sub-family A and B protein functions, including their contribution to antigenic variation and immune evasion.


Subject(s)
Gene Expression Profiling/methods , Genes, Protozoan/genetics , Multigene Family/genetics , Plasmodium chabaudi/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Computational Biology , Conserved Sequence , Female , Host-Pathogen Interactions/genetics , Life Cycle Stages/genetics , Mice , Oligonucleotide Array Sequence Analysis , Plasmodium chabaudi/growth & development , Plasmodium chabaudi/pathogenicity , Plasmodium chabaudi/physiology , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Protozoan/genetics , Sequence Analysis, RNA , Sequence Homology, Amino Acid
20.
Am Nat ; 177(3): 358-67, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21460544

ABSTRACT

All organisms must trade off resource allocation between different life processes that determine their survival and reproduction. Malaria parasites replicate asexually in the host but must produce sexual stages to transmit between hosts. Because different specialized stages are required for these functions, the division of resources between these life-history components is a key problem for natural selection to solve. Despite the medical and economic importance of these parasites, their reproductive strategies remain poorly understood and often seem counterintuitive. Here, we tested recent theory predicting that in-host competition shapes how parasites trade off investment in in-host replication relative to between-host transmission. We demonstrate, across several genotypes, that Plasmodium chabaudi parasites detect the presence of competing genotypes and facultatively respond by reducing their investment in sexual stages in the manner predicted to maximize their competitive ability. Furthermore, we show that genotypes adjust their allocation to sexual stages in line with the availability of exploitable red blood cell resources. Our findings are predicted by evolutionary theory developed to explain life-history trade-offs in more traditionally studied multicellular taxa and suggest that the answer to the long-standing question of why so few transmission stages are produced is that in most natural infections heavy investment in reproduction may compromise in-host survival.


Subject(s)
Biological Evolution , Competitive Behavior , Malaria/parasitology , Malaria/transmission , Microbial Interactions , Plasmodium chabaudi/genetics , Plasmodium chabaudi/physiology , Animals , Gametogenesis , Genetic Variation , Genotype , Male , Metamorphosis, Biological , Mice , Phenotype , Plasmodium chabaudi/growth & development , Reproduction
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